Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses

Figures

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  FIGURE 1. Schematic structure of the recombinant adenoviral genomes. (A) Recombinant pAd5[P] carrying the foot-and-mouth disease virus (FMDV) P12A3C cassette from O1/Campos strain (brown box) under the control of the cytomegalovirus enhancer-promoter (pCMV) in an E1/E3-deleted Ad5 backbone (Ad5ΔE1–E3). pA, SV40 poly(A) signal; ITR, inverted terminal repeats. (B) Recombinant pAd5[PVP2]OP. This Ad5 genome contains the expression unit at the E1 insertion site in a leftward orientation (3′→5′) in the ΔE1–E3 Ad5 backbone. The expression unit contains the FMDV P12A3C cassette under the control of the optimized pCMV (pCMV and human β-globin/IgG chimeric intron, zigzag line). Additionally, it harbors two non-synonymous substitutions in the VP2 coding sequence to express a S93F/Y98F VP2 mutant protein. (C) Recombinant pAd5[P]OP is identical to pAd5[PVP2]OP, but expresses a native VP2 capsid protein.

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  FIGURE 2. Rational design to produce empty capsids of foot-and-mouth disease virus (FMDV) O1/Campos. (A) Scheme of the icosahedral FMDV capsid. A protomer consisting of VP1, VP2, VP3, and the internal polypeptide VP4 (not visible) is shown in orange. The interface between two pentamers at the two-fold symmetry axis, where two neighboring VP2–VP2 interact, is enclosed within a dashed rectangle. (B) The VP2 structure of FMDV O1/Campos was modeled as described in the section “Materials and Methods”. This model led to the prediction and potential localization of residues S93 and Y98, located on the α-helix A (amino acids K88–Y98) at the two-fold symmetry axis.

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  FIGURE 3. Analysis of foot-and-mouth disease virus (FMDV) capsid protein expression. Lysates of human embryonic kidney (HEK) 293A cells infected with Ad5[P], Ad5[P]OP, or Ad5[PVP2]OP were analyzed by SDS-PAGE and Western blot using FMDV anti-O1/Campos polyclonal serum (A). An Ad5 encoding an irrelevant antigen (green fluorescent protein), Ad5[G], was also included. To estimate FMDV structural protein accumulation, the ratios of VP0 to β-tubulin bands were calculated and normalized to the Ad5[P] VP0/β-tubulin ratio, set at 1.0. Mean values from two independent experiments are presented. Standard deviations ranged from 0% to 2% (not shown). VP0, VP1/VP3, and β-tubulin are indicated with an arrow. (B) Analysis of 3C mRNA expression in HEK 293A cells infected with Ad5[G], Ad5[P], Ad5[P]OP, or Ad5[PVP2]OP by quantitative real-time PCR. HEK 293A cells were infected with the corresponding Ad5 vector at multiplicity of infection (MOI) 5. Twenty-four hours post-infection, total RNA was isolated and used to amplify a fragment of the FMDV 3Cpro open reading frame (ORF) and, as a control, a fragment of human Actin ORF by RT-qPCR. For each experimental group, the FMDV 3Cpro transcript level relative to…

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  FIGURE 4. Assembly of recombinant capsid proteins into subviral particles. (A) Lysates of HEK 293A cells infected with Ad5[P], Ad5[P]OP, or Ad5[PVP2]OP were loaded onto 10–45% sucrose gradients. Inactivated native A/Argentina/2001 foot-and-mouth disease virus (iFMDV A/Arg/01) was also included as a marker (black dotted line). Fractions were collected after centrifugation at high speed and analyzed by ELISA. The positions of the FMDV virions (146S), empty capsids (75S), and capsomers (12S) are indicated. (B) Eight fractions (5, 10, 15, 20, 25, 30, 35, and 40) were analyzed by Western blot with specific anti-FMDV O1/Campos polyclonal antibody. VP0 and VP1/VP3 are indicated with an arrow. (C) Negative-staining TEM of in situ generated FMDV empty capsid particles (right) and iFMDV 146S and 75S particles (left). Scale bar, 50 nm. Virus-like particles are indicated (filled inverted triangle).

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  FIGURE 5. Comparative detection of foot-and-mouth disease virus (FMDV) transcripts in draining popliteal lymph nodes (dPLNs) of inoculated mice. Comparison of transcript accumulation levels in dPLNs after inoculation of BALB/cJ mice. Animals (n = 3) were inoculated i.m. with 5 × 108 PFU of Ad5[G], Ad5[P], or Ad5[PVP2]OP. Total RNA was extracted from dPLNs at 2 and 8 dpi, respectively, and a fragment of the FMDV 3Cpro open reading frame (ORF) and the GAPDH ORF were amplified by RT-qPCR. For each experimental group, the FMDV 3Cpro transcript level relative to the Ad5[P] group was calculated by using the 2− ΔΔCt equation. Data correspond to the average and standard deviation from two independent experiments. *p < 0.01 (ANOVA).

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  FIGURE 6. Immune responses in mice after the administration of Ad5[P]OP and Ad5[PVP2]OP particles. (A) Kinetics of foot-and-mouth disease (FMD) antigen-specific antibodies in BALB/cJ mice. The syringe indicates the booster inoculation (at 28 dpi). Asterisks indicate statistical significance in antibody titers (p < 0.05). FMD virus (FMDV)-specific antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) in sera from animals bled at 35 and 45 days post-inoculation. (B) Isotype antibody profile induced by different Ad5 candidates. ELISA titers of anti-FMDV-specific IgG1 or IgG2a determined at 45 dpi are shown. Asterisk indicates the statistically significant difference between the IgG1 and IgG2a levels (p < 0.05). (C) IgG2a/IgG1 ratio measured in each experimental group. The consistency of the IgG ratios was assessed through the determination of the significant differences in the titers of both isotypes in each group (shown at the top of the bars). (D) Avidity of the antibodies induced by different immunogens. Serum samples from inoculated mice at 45 dpi were used to estimate the avidity index as a percentage. The average index corresponded to the optical density (OD) 405…

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  FIGURE 7. Protection against challenge with infectious foot-and-mouth disease virus (FMDV) O1/Campos. (A) Protection from viral challenge elicited by the different Ad5-FMD vectors. Groups of mice were inoculated with Ad5[P], Ad5[P]OP, Ad5[PVP2]OP, Ad5[G], or a commercial FMD vaccine and challenged with infective FMDV at 49 dpi. Animals were considered protected if viremia was absent after a blind passage. Protection percentages were calculated as 100 × (number of vaccinated animals without viremia/number of vaccinated animals). (B) Virus neutralization test (VNT) at day 45 post-inoculation with 5 × 108 PFU, expressed as log10 of the reciprocal of the serum dilution that neutralizes 50% of 100 TCID50 of infective FMDV O1/Campos (fixed virus–variable serum method). *p < 0.05, **p < 0.01 with respect to the Ad5[P] group. A serum pool was made every two animals of a group in order to perform the serum neutralization test.

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  FIGURE 8. Bone marrow dendritic cell (BMDC) activation induced by Ad5[P] or Ad5[PVP2]OP infection. BMDCs were infected with Ad5[P] or Ad5[PVP2]OP at multiplicity of infection (MOI) of 50 and 250, as described in the section “Materials and Methods.” Then, the cells were stained with specific monoclonal antibodies (mAbs) for phenotypic and activation markers and analyzed by flow cytometry. (A) Representative histograms depict the fluorescence intensities of CD80, CD86, and major histocompatibility complex (MHC) class II molecules on MHCII+CD11c+ gated population. (B) Bars represent the geometric mean of fluorescence intensity (MFI) ± SD. Isotype, cells were stained with control isotype mAbs; LPS-stimulated, cells were treated with 500 ng/ml lipopolysaccharide (LPS). *p < 0.05, **p < 0.01, ***p < 0.001 compared to non-stimulated cells (ANOVA).